Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

Structure and binding properties of hen lysozyme modified at tryptophan 62

The structure of a derivative of hen egg-white lysozyme (EC 3.2.1.17) modified by N-bromosuccinimide at Trp62 has been studied by both ¹H nuclear magnetic resonance spectroscopy and X-ray crystallography. It was shown that this modification, changing the tryptophan residue to an oxindolealanine² residue, only causes minor structural changes at the site of the modification, and that the overall structure of the native enzyme is maintained in the derivative. Both diastereomers of the oxindolealanine-62 lysozyme were observed by the two methods employed, in accordance with previous observations (Norton & Allerhand, 1976). The pK values of the catalytically important carboxyl groups of Glu35 and Asp52 were identical in the native enzyme and its derivative. However, the modified enzyme is virtually inactive in the hydrolysis of the cell-wall mucopolysaccharide of Micrococcus lysodeikticus. The binding of N-acetylglucosamine oligosaccharides to both native lysozyme and Ox-62 lysozyme was studied by nuclear magnetic resonance spectroscopy, observing the perturbations on the lysozyme ¹H n.m.r. resonances, and differences in the perturbations of the two systems demonstrated that binding of (GlcNAc)₃ in particular was not identical in the two systems. The structure of Ox-62 lysozyme-(GlcNAc)₃ was studied by X-ray crystallography and it was shown that only two GlcNAc residues make contact with the enzyme, binding the reducing end residue in a similar mode as the α-anomeric form of GlcNAc binds to the native enzyme (Blake et al., 1967a). On the basis of the results obtained by X-ray crystallography and ¹H n.m.r. spectroscopy, the lack of enzymatic activity of the Ox-62 lysozyme arises from the obstruction by the oxindolealanine residue of sub-site B of the active site, preventing productive binding of the substrate.     

Treatment of peptidoglycan monomer with aqueous ammonia: Formation of lactoylpeptide and a saturated disaccharide

Treatment of peptidoglycan monomer (1) from Brevibacterium divaricatum with aqueous ammonia led to cleavage of the C-3 ether linkage in the N-acetylmuramoyl residue to give the d-lactoylpentapeptide and a saturated disaccharide. By using ¹³C-n.m.r. spectroscopy, the disaccharide was identified as chitobiosamine. Alkaline treatment of model compounds under similar conditions showed that N-acetylmuramoyl derivatives with C-1 unsubstituted undergo cleavage at C-3 to give the corresponding 2-acetamido-2-deoxy-d-glucopyranose derivative. The reaction of 1 with ammonia was monitored by ¹H-n.m.r. spectroscopy and, from the data obtained, rate constants and the activation energy were calculated.     

Assessing the Soil Carbon,Biomass Production,and Nitrous Oxide Emission Impact of Corn Stover Management for Bioenergy Feedstock Production Using DAYCENT

Harvesting crop residue needs to be managed to protect agroecosystem health and productivity. DAYCENT, a process-based modeling tool, may be suited to accommodate region-specific factors and provide regional predictions for a broad array of agroecosystem impacts associated with corn stover harvest. Grain yield, soil C, and N₂O emission data collected at Corn Stover Regional Partnership experimental sites were used to test DAYCENT performance modeling the impacts of corn stover removal. DAYCENT estimations of stover yields were correlated and reasonably accurate (adjusted r ²?=?0.53, slope?=?1.18, p?<<?0.001, intercept?=?0.36, p?=?0.11). Measured and simulated average grain yields across sites did not differ as a function of residue removal, but the model tended to underestimate average measured grain yields. Modeled and measured soil organic carbon (SOC) change for all sites were correlated (adjusted r ²?=?0.54, p?<<?0.001), but DAYCENT overestimated SOC loss with conventional tillage. Simulated and measured SOC change did not vary by residue removal rate. DAYCENT simulated annual N₂O flux more accurately at low rates (≤2-kg N₂O-N ha^?1 year^?1) but underestimated when emission rates were >3-kg N₂O-N ha^?1 year^?1. Overall, DAYCENT performed well at simulating stover yields and low N₂O emission rates, reasonably well when simulating the effects of management practices on average grain yields and SOC change, and poorly when estimating high N₂O emissions. These biases should be considered when DAYCENT is used as a decision support tool for recommending sustainable corn stover removal practices to advance bioenergy industry based on corn stover feedstock material.     

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-β-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two l-glutamine residues. A substance P derivative with an N-acetyl-d-glucosamine residue attached to the fifth or sixth l-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-d-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the l-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the l-glutamine residue of the peptide was not liberated by peptide-N⁴-(N-acetyl-β-d-glucosaminyl) asparagine amidase F.     

Spliceosome Sm proteins D1, D3, and B/B' are asymmetrically dimethylated at arginine residues in the nucleus

We report a novel modification of spliceosome proteins Sm D1, Sm D3, and Sm B/B′. L292 mouse fibroblasts were labeled in vivo with [³H]methionine. Sm D1, Sm D3, and Sm B/B′ were purified from either nuclear extracts, cytosolic extracts or a cytosolic 6S complex by immunoprecipitation of the Sm protein-containing complexes and then separation by electrophoresis on a polyacrylamide gel containing urea. The isolated Sm D1, Sm D3 or Sm B/B′ proteins were hydrolyzed to amino acids and the products were analyzed by high-resolution cation exchange chromatography. Sm D1, Sm D3, and Sm B/B′ isolated from nuclear fractions were all found to contain ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine, modifications that have been previously described. In addition, Sm D1, Sm D3, and Sm B/B′ were also found to contain asymmetric ω-N^G,N^G-dimethylarginine in these nuclear fractions. Analysis of Sm B/B′ from cytosolic fractions and Sm B/B′ and Sm D1 from cytosolic 6S complexes showed only the presence of ω-N^G-monomethylarginine and symmetric ω-N^G,N^G′-dimethylarginine. These results indicate that Sm D1, Sm D3, and Sm B/B′ are asymmetrically dimethylated and that these modified proteins are located in the nucleus. In reactions in which Sm D1 or Sm D3 was methylated in vitro with a hemagglutinin-tagged PRMT5 purified from HeLa cells, we detected both symmetric ω-N^G,N^G′-dimethylarginine and asymmetric ω-N^G,N^G-dimethylarginine when reactions were done in a Tris/HCl buffer, but only detected symmetric ω-N^G,N^G′-dimethylarginine when a sodium phosphate buffer was used. These results suggest that the activity responsible for the formation of asymmetric dimethylated arginine residues in Sm proteins is either PRMT5 or a protein associated with it in the immunoprecipitated complex.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

Biological and biochemical consequences of the human ERCC-1 repair gene after transfection into a repair-deficient CHO cell line

The consequences of the presence of the human gene ERCC1 in repair-deficient 43-3B cells were examined. The gene restores the sensitivity of this mutant not only to UV but also to 4NQO, N-Ac-AAF and alkylating agents to the normal level. Also, the frequency of mutation induction by UV at the Na⁺/K⁺-ATPase locus returns to the level of CHO wild-type cells. Additionally, the rate of cyclobutane pyrimidine dimer removal approaches that in wild-type CHO cells. The results obtained indicate that the human gene ERCC-1 restores the impaired functions in 43-3B, and that the gene is probably functionally homologous to the defective one in the 43-3B cell line. Some evidence was found for a difference between the human gene product and its rodent counterpart, as the restoration of normal sensitivity to 4NQO, ENU and N-Ac-AAF was complete whereas it was not for UV.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

Substrate specificity of mammalian endo-beta-N-acetylglucosaminidase: study with the enzyme of rat liver

The substrate specificity of mammalian endo-β-N-acetylglucosaminidase was studied in detail by using rat liver enzyme. The enzyme hydrolytically cleaves the N,N′-diacetylchitobiose moiety of Manα1 → 6 (Manα1 → 3)Manβ1 → 4GlcNacβ1 → 4R in which R represents either GlcNac → Asn or N-acetylglucosamine. The enzyme can hardly act on the sugar chains with Fucα1 → 3 or 6GlcNac → Asn or N-acetylglucosaminitol as their R residues. The sugar chains substituted at C-3 and C-6 positions of the Manα1 → 6 residue and at C-2 position of the Manα1 → 3 residue by other sugars are also cleaved by the enzyme. The sugar chains substituted at C-4 position of the β-mannosyl residue and at C-2 position of the Manα1 → 6 residue by other sugars are hydrolyzed at one place lower rate. The specificity of the mammalian endo-β-N-acetylglucosaminidase indicates that the enzyme is responsible for the formation of most of the oligosaccharides excreted in the urine of patients with congenital exoglycosidase deficiencies and also explains why large amount of glycopeptides are excreted in the urine of fucosidosis patients.     

Removal of the N-terminal residue of a protein after transamination

1. Pseudomonas cytochrome c-551 was modified by treatment at 20° with glyoxylate in the presence of pyridine and cupric sulphate. The change in its chromatographic properties was consistent with conversion of its N-terminal residue into an oxo acyl residue by transamination. 2. The product underwent further modification on treatment with o-phenylenediamine or 4-methylphenylene-1,2-diamine in strong acetate buffer at 37°. The final product had chromatographic properties and the N-terminal residue consistent with its differing from the native cytochrome solely in the absence of the original terminal residue. 3. The nature of analogous reactions supports these interpretations of the modifications. 4. These two treatments provide a method for specific removal of the N-terminal residue of a protein. 5. The intermediate and final products were oxidized by cytochrome oxidase at about the same rate as the original cytochrome.     

Teichoic acids of three type strains of the Bacillus subtilis group, Bacillus mojavensis VKM B-2650, Bacillus amyloliquefaciens subsp. amyloliquefaciens VKM B-2582, and Bacillus sonorensis VKM B-2652

Cell walls of three type strains of the Bacillus subtilis group, Bacillus mojavensis VKM B-2650, Bacillus amyloliquefaciens subsp. amyloliquefaciens VKM B-2582, and Bacillus sonorensis VKM B-2652, are characterized by the individual set of teichoic acids. All strains contained 1,3-poly(glycerol phosphates), unsubstituted, acylated with D-alanine, and glycosylated. The latter differ in the nature of the monosaccharide residue. Teichoic acids of B. mojavensis VKM B-2650^T and B. amyloliquefaciens subsp. amyloliquefaciens VKM B-2582^T contained α-glucopyranose, while those of B. sonorensis VKM B-2652^T contained β-glucopyranose and N-acetyl-α-D-glucosamine. Moreover, cell walls of B. mojavensis VKM B-2650^T contained a teichoic acid of poly(glycosylglycerol phosphate) nature with the following structure of the repeating unit: -4)-α-D-α-D-GlcpNAc-(1 → 3)]-Glcp-(1 → 2)-sn-Gro-(3-P-. The type strains have been characterized according to the composition of cell wall sugars and polyols. Application of teichoic acids (set and structure) as chemotaxonomic characteristics is discussed for six type strains of the Bacillus subtilis group. Polymer structures were determined by chemical and NMR spectroscopic techniques.     

Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides

Acylated crude papain has been shown to exert stereoselective behavior toward racemic hydrazides devoid of an amino acid residue, namely, (RS)-mandelic and (RS)-atrolactic hydrazides. These hydrazides functioned as nucleophiles to yield N¹,N²-diacylhydrazines. Several achiral acylating agents for the enzyme were chosen, including Z-glycine, BOC-glycine, AOC-glycine, and hippuric acid. With the exception of hippuric acid as the acylating agent, the reaction product, in every instance for these achiral hydrazides, consisted of an excess of the (+)-N¹,N²-diacylhydrazine. The relative rates of product formation for the mandelic hydrazides were considerably greater than for corresponding reactions with racemic atrolactic hydrazide. When chiral Z-l-alanine was employed to acylate crude papain, the stereoselective action was most pronounced, with the formation of a mixture of diastereoisomers consisting of 73% N¹-(Z-l-alanyl)-N²-[(R)-mandelyl]hydrazine. The relative reactivities for the electrophiles was Z-l-alanine ? Z-glycine ? hippuric acid ? AOC-glycine > BOC-glycine. The hydrazides of (R)-, (S)-mandelic, and (RS)-atrolactic acids were prepared by conversion of the corresponding acids to their esters by means of a catalytic dehydrating agent and subsequent treatment with a methanolic solution of hydrazine.     

A rat kidney neutral peptidase that degrades B chain of insulin, glucagon, and ACTH: purification by affinity chromatography and some properties.

A metallo-endopeptidase that catalyzes at near neutral pH the hydrolysis of certain polypeptides was purified from rat kidney microsomes by a simplified procedure using affinity chromatography on Sepharose 4B coupled with insulin B chain. The purified enzyme showed a single component by chromatography on diethylaminoethyl cellulose and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 213,000. Studies on its substrate specificity showed that the purified enzyme rapidly degrades insulin B chain, glucagon, adrenocorticotropin, and, at a significantly lower rate, insulin A chain. The enzyme has a very weak or no activity toward ribonuclease and vasopressin. In contrast, the enzyme does not degrade denatured hemoglobin, bovine serum albumin, insulin (nano- or micromolar), oxytocin, furylacryloylglycyl-leucine amide (FAGLA), synthetic substrates of cathepsin C (β-napthalamides of glycine-l-arginine and l-histidine-l-serine), or synthetic substrates of aminopeptidases (l-arginine- or l-glutamic acid-β-napthylamide). The enzyme degrades reduced or oxidized B chain at about the same rate, but S-sulfonated B chain is degraded at a markedly lower rate. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. Activity of the enzyme is markedly inhibited by chelating agents (EDTA and o-phenanthroline) and, modestly, by high concentrations of citrate and histidine. Activity of the enzyme is also markedly inhibited by simple thiol compounds (dithiothreitol, glutathione, and mercaptoethanol), but not by sulfhydryl reagents (N-ethylmaleimide or iodoacetate). The inactive apoenzyme, prepared by treatment of the enzyme with EDTA followed by dialysis, was reactivated by Zn²⁺ > Ca²⁺, minimally by Cu²⁺, but not by Hg²⁺. Some anions (phosphate, borate, and bicarbonate) were strongly inhibitory, but chloride had no effect. The following agents were found to have no effect: soybean and lima bean trypsin inhibitors, N^?-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N^α,?-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin (Trasylol), phenylmethylsulfonyl fluoride (a serine protease inhibitor), 1-methyl histidine, 3-methyl histidine, histamine, imidazole, and heparin.     

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N⁶-ethenoadenine (ɛA), 3,N⁴-ethenocytosine (ɛC) and 1,N²-ethenoguanine (1,N²-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate.Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N²-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N²-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.     

Semisynthetic analogues of insulin. The use of N-substituted derivatives of methionine as acid-stable protecting groups

1. We describe the use of benzyloxycarbonylmethionine and ethoxycarbonylmethionine for the selective protection of the amino groups of glycine-A1 and lysine-B29 of pig insulin. We have used the Edman method to remove residues from the N-terminal and of the B-chain of the N^A1N^B29-di-protected derivatives. The benzyloxycarbonyl group shows slight but noticeable lability in the acid-cleavage step, but the ethoxycarbonyl group remained intact even after five cycles of degradation. 2. We have prepared the following truncated forms of insulin via the di(ethoxycarbonylmethionyl) derivative: des-Phe^B1-insulin;des-(Phe^B1-Val^B2)-insulin; des-(Phe^B1-Val^B2-Asn^B3)-insulin;des- (Phe^B1-Val^B2-Asn^B3-Gln^B4)-insulin; des-(Phe^B1-Val^B2-Asn^B3 -Gln^B4-His^B5)-insulin. 3. Insulin was re-synthesized from the di-protected des-Phe^B1-insulin by reaction with an active ester of t-butoxycarbonyl-l-phenylalanine. The product after deprotection crystallized, and the immunoreactivity of the crystalline material was identical with that of the native protein. 4. We have prepared the following analogues of insulin in a similar manner: [l-Ala^B1]insulin; [l-Val^B1]insulin; [l-Tyr^B1]insulin; [m-F-l-Phe^B1]insulin; [o-F-l-Phe^B1]-insulin; [o-F-l-Phe^B2]des-Phe^B1-insulin. All had between 34 and 62% of the activity of insulin in the fat-cell test. 5. We have also investigated the use of the benzyol, toluene-p-sulphonyl, p-nitrobenzyloxycarbonyl and 2,4-dinitrophenyl groups for the N-protection of the methionine active esters. Each should have had some particular advantage over the benzyloxycarbonyl and ethoxycarbonyl groups, but all proved in practice to have disadvantages that more than outweighed anything in their favour.     

New archaeal methyltransferases forming 1-methyladenosine or 1-methyladenosine and 1-methylguanosine at position 9 of tRNA

Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N¹-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N¹ atom of a purine residue is discussed.     

Expression of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (Total) and pCyclin B1Ser126 in Vulvar Squamous Cell Carcinoma and Their Relations with Clinicopatological Features and Prognosis

Cyclin B1-CDK1 complex plays an important role in the regulation of cell cycle. Activation of Cyclin B1 and CDK1 and the formation of the complex in G2/M are under multiple regulations involving many regulators such as isoforms of 14-3-3 and CDC25 and Wee1. Abnormal expression of Cyclin B1 and CDK1 has been detected in various tumors. However, to our knowledge no previous study has investigated Cyclin B1 and CDK1 in vulvar cancer. Therefore, we evaluated the statuses of CDK1^Tyr15, pCDK1^Thr161, Cyclin B1 (total) and pCyclin B1^Ser126 in 297 cases of vulvar squamous cell carcinomas by immunohistochemistry. Statistical analyses were performed to explore their clinicopathological and prognostic values. In at least 25% of tumor cases high expression of CDK1^Tyr15, pCDK1^Thr161, Cyclin B1 (total) and pCyclin B1^Ser126 was observed, compared to the low levels in normal vulvar squamous epithelium. Elevated levels of CDK1^Tyr15, pCDK1^Thr161, Cyclin B1 (total) and pCyclin B1^Ser126 were correlated with advanced tumor behaviors and aggressive features. Although CDK1^Tyr15, pCDK1^Thr161, Cyclin B1 (total) and pCyclin B1^Ser126 could not be identified as prognostic factors, combinations of (pCDK1^{Thr161 C+N} + 14-3-3σ^N), (pCDK1^{Thr161 C+N} + 14-3-3η^C), (pCDK1^{Thr161 C+N} + Wee1^C) and (pCDK1^{Thr161 C+N} + 14-3-3σ^N + 14-3-3η^C + Wee1^C) were correlated with disease-specific survival (p = 0.036, p = 0.029, p = 0.042 and p = 0.007, respectively) in univariate analysis. The independent prognostic significance of (pCDK1^{Thr161 C+N} + 14-3-3σ^N + 14-3-3η^C + Wee1^C) was confirmed by multivariate analysis. In conclusion, CDK1^Tyr15, pCDK1^Thr161, Cyclin B1 (total) and pCyclin B1^Ser126 may be involved in progression of vulvar squamous cell carcinoma. The combination of pCDK1^Thr161, 14-3-3σ, 14-3-3η and Wee1 was a statistically independent prognostic factor.